The present invention relates to a method for preventing the denaturation of blood, particularly hemoglobin, and a blood test tool to be used therein.
Hemoglobin Alc (HbAlc) in which hemoglobin (Hb) is bonded to glucose reflects the average glucose level of the body one or two months earlier. Therefore, it is widely used in physical examinations for geriatric diseases or treatment and consultation therefor. However, since it takes a long time to measure HbAlc, for example, even if the blood is collected from outpatients when they visits to hospital, the result of the examination of HbAlc is generally evaluated when the patient visits to the hospital the next time. Therefore, although the measurement value of HbAlc is an important factor for diagnosis of diabetes, actually, it has not been used effectively for treatment.
To solve this problem, a blood collection card made of filter paper has been proposed (xe2x80x9cDiabetesxe2x80x9d Vol. 38, No. 10 (1995), JP10-104226A, etc.). The patients collect blood onto the collection card by themselves and allow the collection card to be impregnated with the collected blood, dried, and then mail the card to the hospital. In the hospital where this card is received, the part impregnated with the blood of the card is cut out or punched out (punch-out), and then blood is eluted therefrom. The eluted blood is examined for the predetermined items including HbAlc, etc. When the patient visits to the hospital, the treatment or diagnosis is performed based on the examination results.
However, one to several days are required for collecting blood onto the collection card, mailing the collection card, and examining the blood. Furthermore, since the blood is held in a dried state in this case, Hb may be denatured. For example, if Hb and glucose react with each other in the blood collection card, and HbAlc is newly produced, the value of HbAlc produced in the body cannot be determined exactly. Furthermore, if the protein-denaturation of Hb occurs, a denatured Hb peak appears in the measurement using the high performance liquid chromatography (HPLC), and a denatured Hb peak appears and affects the peak of HbAlc, which may lead to the inability to measure or measurement errors.
It is therefore an object of the preset invention to provide a method for preventing the denaturation of blood in a dried state and the blood test tool for using the method.
In order to attain the above-mentioned object, the present invention provides a method for preventing the denaturation of blood in a dried state, wherein the blood in a dried state and a carboxylic acid are allowed to be present together
When carboxylic acid and blood are allowed to be present together, even in a dried state, the production of new HbAlc is prevented and also the protein-denaturation of Hb is prevented. In the production of HbAlc, first Hb and glucose are reacted nonenzymatically with each other (a reversible reaction) to produce an unstable aldoimine, which is formed into a stable ketoamine (HbAlc) through an Amadori rearrangement. Carboxylic acid is believed to prevent the production of the unstable aldimine so as to prevent the production of new HbAlc. On the other hand, a mechanism of preventing the protein-denaturation of Hb is not clear. However, as known from the below mentioned Examples, the effect is significant.
Furthermore, in the present invention, xe2x80x9cHb is denaturedxe2x80x9d means not only that the structure of the protein is changed like protein-denaturation, but also modification of Hb by other materials, and the like.
It is preferable in the method for preventing the denaturation of blood of the present invention that carboxylic acid is added to blood in a non-dry state and then the blood is dried. In this way, if carboxylic acid is added to a liquid blood before being dried, since the blood and carboxylic acid can be brought into contact with each other sufficiently, it is possible further to prevent the denaturation of the blood in the following dried state.
It is preferable in the present invention that the carboxylic acid is citric acid because it exhibits a high effect in preventing the denaturation of Hb. However, in the present invention, as the carboxylic acid, for example, oxalic acid, pyruvic acid, acetic acid, etc. may be used. The amount of carboxylic acid to be added is generally 1.5 to 10 parts by weight, and preferably 2.5 to 6 parts by weight, with respect to 100 parts by weight of the blood in a non-dry state.
It is preferable in the method for preventing the blood denaturation of the present invention that in addition to carboxylic acid, a non-reducing sugar and the blood are allowed to be present together because the non-reducing sugar is not likely to be reacted with Hb and can prevent the denaturation of Hb further. It is preferable that the non-reducing sugar is sucrose. Besides, a non-reducing sugar such as trehalose can be used. The amount of the non-reducing sugar is generally 10 to 50 parts by weight, and preferably 20 to 30 parts by weight, with respect to 100 parts by weight of the blood in a non-dry state.
It is preferable in the method for preventing the blood denaturation of the present invention that, in addition to the carboxylic acid and the non-reducing sugar, at least one of an anticoagulant and an antioxidant is allowed to be present together with blood. An example of the anticoagulant includes ethylenediaminetetraacetic acid (EDTA), heparin, oxalic acid salt, and the like. Among them, EDTA is preferable. The amount of the anticoagulant is generally 1 to 5 parts by weight, and preferably 1.5 to 3 parts by weight, with respect to 100 parts by weight of the blood in a non-dry state. An example of the antioxidant includes glutathione (GSH), xcex1-tocopherol, and the like. Among them, GSH is preferable. The amount of the antioxidant is generally 0.1 to 8 parts by weight, and preferably 0.1 to 3 parts by weight, with respect to 100 parts by weight of the blood in a non-dry state.
It is preferable in the method for preventing the blood denaturation of the present invention that the blood is in a dried state, with a porous material being impregnated with the blood and the blood dried.
Next, according to the blood test tool of the present invention, the blood test tool holds blood in a dried state with a porous material impregnated with the blood, and a part for holding the blood of the porous material (hereinafter, xe2x80x9cblood holding partxe2x80x9d will be referred to) contains carboxylic acid.
In the blood test tool of the present invention, like in the method for storing the blood of the present invention, it is preferable that the carboxylic acid is citric acid. The above-mentioned carboxylic acids may be used. Furthermore, the amount of carboxylic acid is the same as in the above.
It is preferable in the blood test tool of the present invention that the blood holding part contains a non-reducing sugar, in addition to carboxylic acid. The non-reducing sugar is preferably sucrose, and the above-mentioned non-reducing sugar can also be used. Furthermore, the amount of the non-reducing sugar is the same as in the above.
It is preferable in the blood test tool of the present invention that the blood holding part contains at least one of an anticoagulant and an antioxidant in addition to carboxylic acid and non-reducing sugar. The anticoagulant is preferably EDTA, and the above-mentioned anticoagulants also can be used. Furthermore, the amount of the anticoagulant is the same as in the above. The above-mentioned antioxidant is preferably GSH, and the above-mentioned antioxidants also can be used. The amount of the antioxidant is the same as in the above.
It is preferable in the blood test tool of the present invention that the blood holding part is surrounded by a blood anti-diffusion layer provided in or around the porous material.
When the blood test tool of the present invention has such a liquid anti-diffusion layer, it is possible to prevent the blood smeared on the porous material from diffusing, so that the diffusion of the blood in the direction of thickness is promoted. Therefore, the blood can be secured in a constant region (the blood holding part) of the porous material. Furthermore, since the blood holding part is the above-mentioned constant region, it is possible to prevent a sieve effect by the porous material. For example, there is little concentration difference in the components such as the hemocytes between the central part to which the blood is smeared and the diffusion part in which the blood is diffused in the planar direction. Thus, it is possible to maintain homogeneously the concentration of the components in blood in the planar direction of the blood holding part. Therefore, for example, the part to be punched out for the blood holding part is not particularly limited, so that the procedure is much simplified, and also measurement can be carried out with excellent repeatability.
It is preferable in the blood test tool of the present invention that the blood anti-diffusion layer has a non-porous structure since it is possible reliably to prevent the blood in the blood holding part from diffusing to the outside.
The layer of the non-porous structure may be formed, for example, by impregnating the porous material with resin so as to make the porous structure into a non-porous structure. An example of the resin with which the porous material is impregnated includes, for example, polyvinyl butyral, ethyl cellulose, cellulose acetate, polyethylene terephthalate, polyethylene, gelatin, and the like.
Furthermore, the liquid anti-diffusion layer may be a member, for example, a plastic such as polystyrene, polyethylene terephthalate, polyvinyl chloride, etc.; glass; metal; and the like. The porous material may be surrounded by these members.
Furthermore, it is preferable that the blood anti-diffusion layer is hydrophobic since it is possible to prevent the blood from diffusing regardless of a non-porous structure and a porous structure. An example of the hydrophobic blood anti-diffusion layer may be a layer formed by impregnating the porous material with hydrophobic resin. An example of the usable hydrophobic resin includes, for example, polyacetal resin, fluororesin, silicone resin, and the like.
It is preferable in the blood test tool of the present invention that the porous material is a card type. The card type blood test tool is excellent in handling property and is convenient when mailed, and the like.
It is preferable in the blood test tool of the present invention that the blood holding part is supported by a supporter and is detachable from the supporter. In this way, if the blood holding part is detachable from the supporter, for example, a step of punching out the card can be omitted and the generation of pieces can be prevented. Therefore, prior to the measurement, it is not necessary to remove pieces by filtration or centrifuge, thus making the measurement more easily.
It is preferable that the blood test tool of the present invention has a cylindrical shape with a bottom and the bottom part is formed of a porous material. When a test tool has such a cylindrical shape with a bottom, the blood test tool itself can be used as a container for elution, and thus it is not necessary to prepare the particular container for elution. Furthermore, for example, after the porous material of the blood test tool is impregnated with blood and the blood is dried, the eluate (extractant) is put into the test tool so as to infiltrate into and permeate the porous material at the bottom part, thereby eluting the blood. Thus, it is possible to elute the blood easily and promptly. Specifically, for example, when the eluate that is put into the cylindrical blood test tool with a bottom infiltrates into the porous material by gravitaty, and when the eluate passes through the porous material, the blood held is eluted (extracted). The eluate passing through the porous material is collected in the container located in the lower part of the blood test tool, whereby the elution is completed. With such a blood test tool, for example, right after the elution is completed, the measurement can be performed, so that the punching-out step can be omitted, and the generation of small pieces of the porous material is prevented. Furthermore, a special machine such as a shaker, etc. is not used and the elution can be performed briefly for a short time.